Respiratory distress syndrome of the newborn (RDS) which is a leading cause of infant mortality and morbidity in the United States is associated, at least in part, with a deficiency in the level of a surfactant complex which normally is present at the air-fluid surface of the alveoli. The alveoli are small sacs in which gases are exchanged between blood and the air spaces of the lung, and the surfactant complex serves to reduce surface tension at this interface. If adequate levels of surfactant are not present, the alveoli will collapse during exhalation and cannot be reinflated by subsequent inhalations. The surfactant is a complex of an apoprotein (alveolar surfactant protein, "ASP") and phospholipids, principally dipalmitylphosphocholine and phosphatidylglycerol, and is apparently stabilized by calcium ion.
Because appropriate levels of the surfactant complex are necessary for proper respiratory function, and because insufficient amounts of the complex are indicators of the presence of the syndrome, assay methods designed to detect the levels of components of this complex are important diagnostics of this syndrome. Because these components are present in the amniotic fluid, methods for their detection can be used in predicting the probability of incidence of this deficiency in premature and certain other infants associated with difficult or complicated pregnancies. This is made feasible by the fact that human fetal lung fluid exchanges freely with the external amniotic fluid; indeed, it has been estimated that 20-40% of the surfactant complex or its components are found in the amniotic fluid. The levels of the surfactant complex in the amniotic fluid are thus a good indicator of lung maturity and the risk of RDS developing at birth.
Presently existing diagnostic methods are, however, unsatisfactory. Assays of amniotic fluid (which can be withdrawn by amniocentesis) are based on measurement of the surfactant-specific phospholipids and the ability of the surfactant to stabilize bubbles at an air-water interface or other measures less closely related to the surfactant complex per se. See, e.g., U.S. Pat. No. 4,233,032 which discloses a surfactant test, and U.S. Pat. No. 4,071,770 which relies on fluorescence polarization. The most common phospholipid measurement is a lecithin/sphingomyelin ratio (L/S ratio), which may also be done in conjunction with determination of saturated phosphatidylcholine (SPC) (Torday, J., et al, New England Journal of Medicine (1979) 301:1013). Since there are other sources of phospholipids besides the surfactant complex, these assays are troubled by a relatively high number of false positives, especially in specimens contaminated with blood or meconium, or with specimens from complicated pregnancies such as those of diabetic mothers. Assays are commercially available which are based on measurement of the functional ability of surfactant to stabilize bubbles. For example, Beckmans' Lumadex.TM. FSI Fetal lung maturity test is one of these. False positives are a major problem with these assays as well.
Immunoassays of the ASP should offer inherently increased specificity and diminution of false positives. A relatively insensitive agglutination assay using polyclonal antiserum was used by King, R. J., et al, J. Appl Physiol (1975) 39:735-741, but was never applied to clinical diagnosis. More sensitive radioimmunoassays have been developed to measure animal ASP, but have also been based on polyclonal antisera (King, R. J., et al, American Review of Respiratory Disease (1974) 110:273). The ELISA type assay disclosed by Katyal, S. L., et al, Am J Obstet Gynecol (1984) 148:48 also uses polyclonal antibodies prepared by injecting human or monkey lung washes into guinea pigs and rabbits. The inherent lack of affinity and specificity for ASP of polyclonal sera acts as a detriment to these assays.
The sensitivity and specificity of the assay would be greatly improved by the availability of homogeneous populations of antibodies directed against specific determinants of the ASP amino acid sequence. The present invention provides a source of such specific and homogeneous preparations.